Proteominer protein enrichment kit

For proteomics analysis, serum samples from 5 rat models in each group were mixed in equal volume to form pooled samples. We used a prepacked affinity LC column (1 mL) for depleting the most common proteins by ProteoMiner^TM Protein Enrichment Kit (Bio-Rad), because the pooled serum samples were enriched with low-abundance proteins; protein depletion was performed according to the manufacturer’s recommended protocol. After enrichment, the protein content of each group was determined using the Bradford method. Moreover, the protein integrity of each group was detected by SDS-PAGE.

The serum samples were removed from high‐abundance proteins using the ProteoMinerTM protein enrichment kit (Bio‐Rad, Shanghai, China), and the protein concentration of the treated serum was determined by a Bradford protein quantitative kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions.

Different mixed samples from the same group (4 samples of NBD-NFPA fibroblasts group and 4 samples of BD-NFPA fibroblasts group) at a ratio of 1:1:1:1, individually, and measured for concentrated low-abundance proteins with ProteoMiner protein enrichment kit (Bio-Rad, California, USA). Total protein was extracted using a protein extraction buffer consisting of 7 M urea, 2 M Thiourea, 4% Chaps, 1% DTT, and 0.5% (v/v) protease inhibitor cocktail. According to the manufacturer’s instructions of TMT Isobaric Mass Tag Labeling kit (Thermo, USA), protein pellets (100 μg of each sample) were resuspended in 100 mM triethylammonium bicarbonate (TEAB) with 200 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and incubated for 1 h at 55 °C. After addition of 5 μL of 375 mM iodoacetamide (IAA), the samples were incubated for 30 min at room temperature in the dark, then digested overnight with trypsin at 37 °C (Promega). Each 100 µg sample was labeled with 41 µl of the TMT Label Reagents following the manufacturer’s protocol. The NBD-NFPA fibroblasts sample was labeled with reporter tag 126, whilst the BD-NFPA fibroblasts sample was labeled with reporter tag 127. Following labeling, the peptide mixtures were pooled and desalinated for LC-MS/MS analysis.

Combinatorial peptide ligand library (CPLL) with ProteoMiner protein enrichment kit (Bio-Rad Laboratories, Hercules, CA) was used to pre-treat plasma samples as previously described [25] (link). These hexapeptides beads bind all the plasma proteins equally and allow to eliminate the excess of unbound majority proteins. The low-abundance plasma proteins are enriched compared to the high-abundance proteins which are in smaller quantities. The dynamic range is thus reduced. Columns containing these beads were previously washed and 200 µL of samples was then added and rotated end to end for 2 h at room temperature. Unbound proteins were then eliminated and beads were washed. Sample was then eluted by adding 20 µL elution reagent, repeated three times. Elution samples obtained from all 3 centrifugations were pooled and stored at −20°C.

The ProteoMiner protein enrichment kit (BIO-RAD, Hercules, CA) was applied to enrich the serum proteins with low abundance according to the manufacturer's instructions. A total of 30 μg proteins from six individual TIF or six enriched serum samples were separated by 12% SDS-PAGE, and the gel bands with molecular weights of 25-30 kDa were dissected into small and uniform pieces, which were then subjected to in-gel tryptic digestion [40 (link)]. Subsequently, the lyophilized peptides were dissolved in 2% acetonitrile with 0.1% aqueous formic acid, and each sample was spiked with β-Galactosidase (BG) peptides at a final concentration of 2 fmol/μL as an internal standard for quality control and the normalization of MRM signals.

Before detection, we depleted the most abundant proteins in all eight serum samples (4 vs 4) by using the ProteoMiner protein enrichment kit (Bio-Rad Laboratories, Inc.). A total of 100 μg of protein from each sample that was 5 times volume diluted with triethylammonium bicarbonate was used for further tryptic digestion. The digestion process was performed by adding trypsin (Promega, Madison, WI, USA) to the samples with an enzyme-protein ratio of 1:50 (w/w), the mixture was then stored at 37 °C overnight. One volume of 0.1% formic acid (FA) solution (enzymatic hydrolysate) was added to acidify the proteins to peptides. Then, the peptides were desalted with a Strata-X C18 column. According to the manufacturer’s protocol, iTRAQ 8-plex kits (AB Sciex Inc., Framingham, MA, USA) were used to label the peptides. Then, a high-performance liquid chromatography (HPLC) system (Thermo DINOEX Ultimate 3000 BioRS) with a Durashell C18 (5 μm, 100 Å, 4.6 × 250 mm) was used to fractionate the labeled samples.